6-Phosphofructo-2-kinase (PFKFB3) promotes cell cycle progression and suppresses apoptosis via Cdk1-mediated phosphorylation of p27 - PubMed (original) (raw)

6-Phosphofructo-2-kinase (PFKFB3) promotes cell cycle progression and suppresses apoptosis via Cdk1-mediated phosphorylation of p27

A Yalcin et al. Cell Death Dis. 2014.

Abstract

The control of glucose metabolism and the cell cycle must be coordinated in order to guarantee sufficient ATP and anabolic substrates at distinct phases of the cell cycle. The family of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1-4) are well established regulators of glucose metabolism via their synthesis of fructose-2,6-bisphosphate (F2,6BP), a potent allosteric activator of 6-phosphofructo-1-kinase (Pfk-1). PFKFB3 is overexpressed in human cancers, regulated by HIF-1α, Akt and PTEN, and required for the survival and growth of multiple cancer types. Although most functional studies of the role of PFKFB3 in cancer progression have invoked its well-recognized function in the regulation of glycolysis, recent observations have established that PFKFB3 also traffics to the nucleus and that its product, F2,6BP, activates cyclin-dependent kinases (Cdks). In particular, F2,6BP stimulates the Cdk-mediated phosphorylation of the Cip/Kip protein p27 (threonine 187), which in turn results in p27's ubiquitination and proteasomal degradation. As p27 is a potent suppressor of the G1/S transition and activator of apoptosis, we hypothesized that the known requirement of PFKFB3 for cell cycle progression and prevention of apoptosis may be partly due to the ability of F2,6BP to activate Cdks. In this study, we demonstrate that siRNA silencing of endogenous PFKFB3 inhibits Cdk1 activity, which in turn stabilizes p27 protein levels causing cell cycle arrest at G1/S and increased apoptosis in HeLa cells. Importantly, we demonstrate that the increase in apoptosis and suppression of the G1/S transition caused by siRNA silencing of PFKFB3 expression is reversed by co-siRNA silencing of p27. Taken together with prior publications, these observations support a model whereby PFKFB3 and F2,6BP function not only as regulators of Pfk-1 but also of Cdk1 activity, and therefore serve to couple glucose metabolism with cell proliferation and survival in transformed cells.

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Figures

Figure 1

Figure 1

Selective inhibition of PFKFB3 reduces F2,6BP, glucose uptake and glycolysis in HeLa cells. HeLa cells were transfected with a control siRNA (Ctrl) or two PFKFB3 siRNA molecules (P31 and P32) and PFKFB3 mRNA by real-time RT-PCR (a), PFKFB3 protein by western blot (b) and densitometry (c), F2,6BP (d), [14C]-2-deoxy-glucose uptake (e) and [3H]-5-glucose utilization (f) were assessed. *_P-_value<0.05

Figure 2

Figure 2

Selective inhibition of PFKFB3 suppresses cell proliferation, cell cycle progression and increases apoptosis by HeLa cells. HeLa cells were transfected with a control siRNA (Ctrl) or two PFKFB3 siRNA molecules (P31 and P32) and effects on viable cell number was assessed at 24–72 h (a), the distribution of the cells in the G1, S and G2 phases of the cell cycle after 48 h by propidium iodide (PI) staining and flow cytometry (b and d) and the presence of PI- and/or Annexin V-positive cells after 72 h by flow cytometry (c and e) were examined. *_P-_value<0.05

Figure 3

Figure 3

Transfection of HeLa cells with PFKFB3 siRNAs reduces nuclear F2,6BP and Cdk1 activity. HeLa cells were transfected with a control siRNA (Ctrl) or two PFKFB3 siRNA molecules (P31 and P32) and, after 48 h, nuclear F2,6BP production was quantified (a) and Cdk1 was immunoprecipitated, incubated with 32P-ATP and recombinant p27, and subjected to SDS-PAGE and western blot analysis (b) and densitometry (c). Protein lysates fractionated from the cytoplasm (C) or the nucleus (N) also were assessed for the presence of Cdk1 phosphoserine substrates by western blot analysis (d). In independent experiments, Cdh1 was immunoprecipitated and analyzed by western blot analysis using antibodies specific for total Cdh1, the Cdk1 substrates PXS*P or S*PXR/K and Oct-1 (e)

Figure 4

Figure 4

p27 protein expression is increased by selective inhibition of PFKFB3. HeLa cells were transfected with a control siRNA (Ctrl) or two PFKFB3 siRNA molecules (P31 and P32) and, after 48 h, Cip/Kip family members p57, p27 and p21 were assessed by western blot analysis (a) and densitometry (b). HCT-116 (c, d) and Panc-1 cells (e, f) were similarly transfected with the two PFKFB3 siRNA molecules (P31 and P32) and examined for p27 protein levels by western blot analysis and densitometry. HeLa cells were also separated into cytoplasmic (C) and nuclear (N) fractions, which were then assessed for PFKFB3, p27, Oct-1 (nuclear control) and a-Tubulin (cytoplasmic control) by western blot analysis (g) and densitometry (h). The Hela cells then were examined for cytoplasmic versus nuclear p27 expression by immunofluorescence using DAPI to localize the nuclei (i)

Figure 5

Figure 5

p27 is required for cell cycle block and apoptosis induced by PFKFB3 inhibition. HeLa cells were transfected with control (Ctrl), PFKFB3 siRNA (P32), p27 siRNA or both and assessed for PFKFB3, p27 or β-Actin expression by western blot analysis (a) and densitometry (b), for cell cycle phase by propidium iodide staining and flow cytometry (c and e) and PI- and/or Annexin V-staining by flow cytometry (d and f) after 48 h

Figure 6

Figure 6

Small molecule inhibition of PFKFB3 increases p27 protein in vitro and in vivo. Lewis lung carcinoma cells were exposed to 3PO and analyzed for p27 expression by western blot (a) and densitometry (b). LLC xenograft-bearing mice were injected intraperitoneally with 3PO (0.07 mg/gm daily) and then euthanized. Three tumors were analyzed per group using immunohistochemistry for p27 (c and d)

Figure 7

Figure 7

PFKFB3 synthesizes F2,6BP, which in turn activates PFK-1 and glycolysis in the cytosol and Cdk1 in the nucleus. Taken together, the data presented in the initial report of the functions of nuclear PFKFB3 and this article support a model whereby PFKFB3 in the cytosol regulates glucose metabolism, whereas PFKFB3 in the nucleus regulates Cdk1 activity and p27 protein levels. Both glucose metabolism and Cdk1 activity are required for growth and resistance to apoptosis

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